Proteomic Analysis of Zymogen Granules

María Gómez-Lázaro; Cornelia Rinn; Miguel Aroso; Francisco Amado; Michael Schrader

Disclosures

Expert Rev Proteomics. 2010;7(5):735-747. 

In This Article

Validation of Proteomics Data & Biological Significance

The recent proteomic studies on ZGs have led to a prominent increase in the number of potential new ZG proteins, especially at the ZGM ( Table 1 ). While increased instrument sensitivity allowed the identification of many more low-abundance proteins, it also uncovered more potential contaminating proteins. Thus, it is important to validate the localization of proteins of interest, which in the case of ZG proteins has mainly been done by antibody-based technologies. In a first approach, isolated ZGs can be subfractionated in a ZGC and ZGM fraction. The latter can be further separated in fractions enriched in either peripheral or integral membrane proteins, and protein distribution can be assessed by immunoblotting, using already known ZG proteins as marker proteins for distribution in the different subfractions.[60,61] As outlined above (see the 'Isolation and subfractionation of ZGs' section), an alternative approach is the application of subtractive proteomics to eliminate putative 'contaminants' and to monitor protein distributions among subcellular fractions.

Further support is obtained by immunofluorescence detection (and quantification of co-localization by pixel-by-pixel analysis of confocal images), which has been performed on pancreatic tissue sections, on isolated acini or on isolated granules, for example, for the confirmation of Rab GTPases or SNARE proteins on ZGs.[34,35,61] Localization on isolated ZGs is advantagous when the protein of interest shows a broader intracellular distribution, for example, cytoplasmic or within the endoplasmic reticulum. In addition, co-localization studies have been performed in granule-containing AR42J cells, an acinar model system.[61] AR42J cells have also been used to monitor the targeting of tagged fusion proteins to granules (e.g., chymase-yellow fluorescent protein).[123] The expression of tagged proteins is useful when specific antibodies are either not available or not applicable for immunocytochemistry. In addition, immunoelectron microscopy has been used to provide further information on protein localization.[32]

As the molecular mechanisms involved in ZG biogenesis, sorting and regulated exocytosis are still poorly defined, there is a critical need for the functional characterization of the new 'players' discovered by proteomic studies, once their identification and localization has been validated. As ZG constituents also play important roles in pancreatic injury and disease,[29,30] further understanding of the key processes in regulated secretion will contribute to the development of new therapeutic options in medicine.[99]

The recent proteomic analyses of purified ZGs and ZGM resulted in the identification of several new 'players' with potential functions in regulated exocytosis and ZG trafficking (e.g., Rab proteins and effectors, and SNARE proteins) as well as in membrane transport ( Table 1 ). In addition, the important presence of proteins known to be localized in other organelles or cell types as presenillins, chymase and ER-resident proteins such as cyclophilin B, were also identified and partially confirmed by different approaches ( Table 1 ).[60,123] There is growing evidence that protein multitasking is a common phenomenon that increases the complexity of proteomic data interpretation.[124] It should also be considered that ZG proteins with a potential function in granule biogenesis, once secreted into the intestinal tract, can fulfil additional, nonrelated functions, for example, in enzyme regulation or host defense. An excellent example is GP2, the major membrane protein of ZGs, which binds to bacterial fimbriae (see the 'Proteomics of ZG proteins in pancreatic juice' section).[25,99] In addition, extracellular chymase has been implicated in regulating the permeability of the intestinal epithelium.[125] It should be noted that ZGs likely represent the transport vehicle for some (but not all) apical proteins, which are targeted to the apical domain of the acinar cells.

Proteomic studies of ZGs largely increased the number of small GTPases (from approximately eight previously known to 23) with a potential function in ZG biogenesis, trafficking and secretion.[21,34,35,60,73] Small G-proteins are supposed to regulate each of the four major steps involved in membrane trafficking: vesicle/granule budding, delivery, tethering and final fusion with the target membrane. The majority of the newly identified small G-proteins have not been subjected to further functional studies, but some have been validated by immunodetection (e.g., Rap1, Rab6 and Rab11A).[34] Most of the Rab proteins described on ZGs are supposed to play a role in the regulation of exocytosis and membrane fusion.[21] Functional studies (including expression of mutated proteins, knockdown by siRNA and generation of knockout mice) have so far been performed on Rab3D, Rab27B, Rab8A and Rap1 (reviewed in [21]). Rab3D and Rab27B, the closest homologues of Rab3, have been implicated in regulating ZG exocytosis.[73,126] Rab27B is supposed to be involved in the tethering of ZGs at the cortical actin web.[34,127] However, studies in Rab3D-deficient mice indicate that Rab3D is not required for exocrine exocytosis but for maintenance of normally sized secretory granules.[122] Rab8A is the only Rab protein so far implicated to act early on in ZG formation.[61] Proteins with a likely role in ZG formation are scarce. So far, only clathrin, adaptor protein complex AP-1 and dynamin (Dyn2) have been identified.[17,18] Silencing of Rab8A (but not Rab3) inhibited granule formation and thus the secretion of zymogens in AR42J cells, and resulted in an accumulation of granule marker proteins within the Golgi complex. Interestingly, it has been demonstrated that Rab8A regulates apical protein localization in intestinal epithelial cells by generating conditional Rab8 knockout mice.[128] The mislocalization and degradation of apical peptidases and transporters in lysosomes was observed, thus leading to a marked reduction in the absorption rate of nutrients in the small intestine, and death. The small intestines of the knockouts were reported to be swollen and to contain undigested milk.[128] However, ultrastructural studies did not reveal alterations in the number of ZGs at postnatal week 2, when controls and knockout mice were compared. Interestingly, a great reduction in ZG number was observed at postnatal week 3, which might be explained by autophagy due to starvation conditions.[Harada A,Pers. Comm. 129]

Knockout mice for some other ZG proteins supposed to participate in the biogenesis or dynamics of the organelle have been generated (e.g., syncollin, GP2, Rab3D, serglycin, muclin and Itmap1).[97,98,101,102,122,130,131] Whereas in some cases, alterations in regulated secretion or an increased susceptibility for pancreatitis were observed, none of the proteins appeared to be essential for ZG biogenesis, pointing to some redundancy in the exocrine secretory system.

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